Microorganism for BBM 928 production

ABSTRACT

The actinomycete &#34;Actinomadura sp.&#34; designated in our culture collection as Strain No. G455-101 has been deposited with the American Type Culture Collection under the Accession Number ATCC No. 31491. The taxonomic description of this microorganism is presented in detail in the specification as are instructions for fermenting aqueous nutrient media with it to produce the BBM 928 antibiotic complex. The BBM 928 complex has utility as an antibacterial agent against gram-positive and acid fast bacteria and exhibits antitumor activity against experimental animal tumors.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of copending application Ser. No. 539,513filed Oct. 10, 1983 and now U.S. Pat. No. 4,631,256 patented Dec. 23,1986. The latter is a division of Ser. No. 390,149 filed June 21, 1982and now U.S. Pat. No. 4,451,456 patented May 29, 1984, and thatapplication is in turn a division of Ser. No. 122,626 filed Mar. 6,1980, and now U.S. Pat. No. 4,360,458 patented Nov. 23, 1982. The latteris a continuation-in-part of application Ser. No. 26,488 filed Apr. 2,1979 and now abandoned. The genealogy may be summarized as follows:

    ______________________________________                                        Serial No.                                                                            Filed         Patented    U.S. Pat. No.                               ______________________________________                                         26,488 April 2, 1979 Abandoned                                               122,626 March 6, 1980 Nov. 23, 1982                                                                             4,360,458                                   390,149 June 21, 1982 May 29, 1984                                                                              4,451,456                                   539,513 Oct. 10, 1983 Dec. 23, 1986                                                                             4,631,256                                   ______________________________________                                    

BACKGROUND OF THE INVENTION

This invention is concerned with a new antitumor antibiotic complex andprocesses for production, recovery and separation into bioactivecomponents.

Based on present spectral data and available physico-chemicalproperties, the antitumor antibiotic complex of the present inventionappears to be structurally related to the quinoxaline group ofantibiotics such as echinomycin, Dell, et al., J. Am. Chem. Soc., 97,2497 (1975), quinomycins, Shoji, et al., J. Antibiotics, 14A, 335(1961), and triostin C, The Merck Index, 9th Ed., 9399. However, theantitumor antibiotic complex is differentiated from these antibiotics inthe following aspects:

1. The instant complex and components thereof contain a quinolinenucleus as a chromophore instead of quinoxaline as in the actinoleukingroup of antibiotics.

2. The instant complex and components thereof do not contain sulfur incontrast to the presence of disulfide or thioacetal bridge in thestructure of actinoleukin antibiotics.

3. The instant complex and components thereof show relatively weakantimicrobial activity compared to the actinoleukins which are potentantibacterial antibiotics.

SUMMARY OF THE INVENTION

A new antitumor antibiotic complex designated BBM-928 is provided by thepresent invention. The complex, which contains at least six components,is prepared by cultivating a BBM-928 producing strain of actinomycetes(ATCC 31491) or a mutant thereof in an aqueous nutrient medium employingsubmerged aerobic conditions. This invention also deals with a processfor recovering the BBM-928 complex from the culture medium andseparation of the complex into its bioactive components bycountercurrent distribution and chromatographic techniques.

The scope of this invention comprehends the BBM-928 complex andbioactive components BBM-928 A, B, C, D, E, and F thereof. It isparticularly concerned with components BBM-928 A, B, C, and D in diluteforms, as true concentrates and in purified form.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is the infrared absorption spectrum of BBM-928A in potassiumbromide.

FIG. 2 is the infrared absorption spectrum of BBM-928A in potassiumbromide.

FIG. 3 is the infrared absorption spectrum of BBM-928C in potassiumbromide.

FIG. 4 is the infrared absorption spectrum of BBM-928D in potassiumbromide.

FIG. 5 shows the proton magnetic resonance spectrum of BBM-928Adissolved in deuterated chloroform using TMS as internal standarddetermined with a NMR spectrometer at a frequency of 90 MHz.

FIG. 6 shows the proton magnetic resonance spectrum of BBM-928Adissolved in deuterated chloroform using TMS as internal standarddetermined with a NMR spectrometer at a frequency of 90 MHz.

FIG. 7 shows the proton magnetic resonance spectrum of BBM-928Cdissolved in deuterated chloroform using TMS as internal standarddetermined with a NMR spectrometer at a frequency of 90 MHz.

FIG. 8 shows the proton magnetic resonance spectrum of BBM-928Ddissolved in deuterated chloroform using TMS as internal standarddetermined with a NMR spectrometer at a frequency of 90 MHz.

DETAILED DESCRIPTION OF THE INVENTION

This invention is concerned with a ne antitumor antibiotic complexarbitrarily designated herein as BBM-928. The complex is believed to bestructurally related to the actinoleukin group of antibiotics and isformed by fermentation of as yet unidentified strain of actinomycetes.Any strain of actinomycetes capable of forming BBM-928 in culture mediummay be used with the preferred producing microorganism designatedActinomadura sp. strain No. B455-101 in the Bristol-Banyu culturecollection. The microorganism was isolated from a soil sample collectedin the Philippine Islands and has been deposited with the American typeculture collection of microorganisms as ATCC 31491.

The novel antitumor antibiotic complex of the instant invention referredto herein has at least six components designated BBM-928A, B, C, D, Eand F. Components A, B, C and D of the BBM-928 complex have beenisolated in crystalline form with chemical structures of components A, Band C identified. The complex (and individual components) of thisinvention have antitumor antibacterial properties. With respect toantibacterial activity, the complex and individual components thereofare useful as nutritional supplements in animal feeds and as therapeuticagents in treating bacterial infection in mammals. Additionally, theantibiotics are useful in cleaning and sterilizing laboratory glasswareand surgical instruments and may be used in combination with soaps,detergents and wash solutions for sanitation purposes. Regardingantitumor effects, the complex and individual components thereof areparticularly useful against a variety of intraperitoneally implantedmouse tumors.

ACTINOMYCETES SPECIES STRAIN NO. G455-101

The following is a general description of the preferred microorganismproducing the antibiotic antitumor complex BBM-928. Observations weremade of the cultural, physiological, and morphological features of theorganism in accordance with standard taxonomic methods, e.g., Shirling,et al., Int. J. Syst. Bacteriol. 16, 313 (1966) and Lechevalier, et al.,Biol. Actinomycetes Related Org. 11, 78 (1976).

Micromorphology

Strain No. G455-101 forms both substrate and aerial mycelia, and thesubstrate mycelium is well-developed, long and branched (0.5-0.8μ inwidth). Distinct fragmentation of the substrate mycelium is not seen.Unlike ordinary species of streptomyces, strain G455-101 bears onlyshort or rudimental aerial mycelia, or does not form any in some agarmedia. Short or long spore-chains are produced in the aerial mycelium,which contains 2-50 oval spores in a chain (mostly 5-20 spores).Spore-chains are straight, flexuous or looped in shape. The spores arespherical (0.3-0.4μ), oval or cylindrical (0.3×1.5-3.0μ) in shape andhave smooth surface. Spores are often separated by empty hyphae. Anamorphous sporangium-like vesicle which envelops short coiledspore-chain is observed occasionally on the aerial mycelium.

Cell Wall Composition and Whole Cell Sugar Components

The cell wall of strain G455-101 contains meso-diaminopimelic acid butlacks glycine. Whole cell hydrolyzate shows the presence of glucose,mannose and madurose (3-0-methyl-D-galactose). The aforementioned cellwall composition and whole cell sugar components indicate that strainG455-101 is an actinomycetes species of cell wall type IIIB.

Cultural and Physiological Characteristics

Strain G455-101 grows abundantly, forms pink or greyish pink aerialmycelium, and produces a reddish water-insoluble pigment in thenutritionally rich agar media, such as yeast extract-malt extract agarand oat meal agar. However, in inorganic salts-starch agar,glycerol-asparagine agar and tyrosine agar, it gives poor growth, formswhite or beige rudimental aerial mycelium, and produces small amount ofreddish pigment. Melanoid pigment is not produced in peptone-yeast-ironagar and tyrosine agar. Nitrate is reduced to nitrite. It growsabundantly at 28° C., 37° C., and 45° C., but does not grow at 10° C. orat 50° C. Pentoses and hexoses are well utilized by the strain. Culturaland physiological characteristics of strain G455-101 are shown in Tables1 and 2, respectively. Utilization of carbon sources is shown in Table3.

                  TABLE 1                                                         ______________________________________                                        Cultural Characteristics of Strain No. G455-101*                              ______________________________________                                        1.  Czapek's agar    G**    no or scant growth                                    (Sucrose-nitrate agar)                                                                         R      dark rose                                                              A      white to pale pink                                                     D      none                                              2.  Tryptone-yeast extract  moderate growth, floccose,                            broth (ISP No. 1)       sedimented and not                                                            pigmented                                         3.  Yeast extract-malt extract                                                                     G      abundant                                              agar (ISP No. 2) R      deep red to reddish brown                                              A      abundant, greyish pink to                                                     purplish pink                                                          D      none                                              4.  Oat meal agar (ISP No. 3)                                                                      G      abundant                                                               R      strong yellowish red                                                   A      moderate, pink                                                         D      greyish yellow                                    5.  Inorganic salts-starch                                                                         G      poor                                                  agar (ISP No. 4) R      light yellowish brown to                                                      dark red                                                               A      scant, white to beige                                                  D      none                                              6.  Glycerol-asparagine agar                                                                       G      poor                                                  (ISP No. 5)      R      yellowish pink to reddish                                                     brown                                                                  A      scant, white                                                           D      none                                              7.  Peptone-yeast extract-iron                                                                     G      poor, plicate                                         agar (ISP No. 6) R      strong reddish orange                                                  A      none                                                                   D      light yellowish orange                            8.  Tyrosine agar (ISP No. 7)                                                                      G      poor                                                                   R      dark red                                                               A      scant, white                                                           D      none                                              9.  Glucose-ammonium salts                                                                         G      poor                                                  agar             R      reddish brown                                                          A      scant, light grey                                                      D      none                                              10. Bennett's agar   G      moderate                                                               R      reddish brown                                                          A      restricted, greyish pink                                               D      none                                              ______________________________________                                         *observed after incubation at 37° C. for 3 weeks.                      **Abbreviation:                                                               G--Growth                                                                     R--Reverse color of substrate mycelium                                        A--Aerial mycelium                                                            D--Diffusible pigment                                                    

                  TABLE 2                                                         ______________________________________                                        Physiological Characteristics of Strain No. G455-101                          Test       Response      Method and Medium                                    ______________________________________                                        Nitrite from                                                                             Positive      Inorganic medium:                                    nitrate                  Czapek's sucrose                                                              nitrate broth                                        Nitrite from                                                                             Positive      Organic medium: 0.5%                                 nitrate                  yeast extract, 1.0%                                                           glucose, 0.5% KNO.sub.3,                                                      0.1% CaCO.sub.3                                      Casein hydrolysis                                                                        Weakly positive                                                                             Luedemann's agar                                     in agar medium           medium                                               Skimmed milk                                                                             Positive                                                           coagulation                                                                   Gelatin lique-                                                                           Negative      15% gelatin in tryptone-                             faction                  yeast extract broth                                                           (ISP No. 1 medium)                                   H.sub.2 S production                                                                     Positive      L-Cysteine (0.1%) added                              from L-cysteine          to tryptone-yeast ex-                                                         tract broth (ISP No. 1                                                        medium) plus agar.                                                            H.sub.2 S detected with a                                                     paper strip containing                                                        10% aq. lead-acetate                                                          solution.                                            Formation of                                                                             Negative      Peptone-yeast-iron agar                              melanoid                 (ISP No. 6) and tyrosine                                                      agar (ISP No. 7).                                    Catalase reaction                                                                        Positive      H.sub.2 O.sub.2 aq. solution                         Oxidase reaction                                                                         Positive      Kovacs' reagent                                      Growth temper-                                                                           Abundant growth                                                                             Bennett's agar                                       ature      at 28, 37 and                                                                 45° C. Poor growth                                                     at 20° C. No growth                                                    at 10° C. and 50° C.                                 ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Utilization of Carbon Sources for Strain No. G455-101                                          PG    Lm                                                     ______________________________________                                        1.     Glycerol        ++      +                                              2.     D(-)-Arabinose  +       +                                              3.     L(+)-Arabinose  +       +                                              4.     D-Xylose        ++      +                                              5.     D-Ribose        ++      +                                              6.     L-Rhamnose      ++      +                                              7.     D-Glucose       +       +                                              8.     D-Galactose     ++      +                                              9.     D-Fructose      ++      +                                              10.    D-mannose       ++      +                                              11.    L(-)-Sorbose    -       -                                              12.    Sucrose         -       -                                              13.    Lactose         -, ± -                                              14.    Cellobiose      +       +                                              15.    Melibiose       -, ± -                                              16.    Trehalose       +       +                                              17.    Raffinose       -       -                                              18.    D(+)-Melezitose -       -                                              19.    Soluble starch  +       -                                              20.    Dulcitol        -       -                                              21.    Inositol        +       -                                              22.    D-Mannitol      ++      +                                              23.    D-Sorbitol      -       -                                              24.    Salicin         -       -                                              25.    Cellulose       +       +                                              26.    Chitin          +       +                                              27.    Keratin         +       +                                              ______________________________________                                         Basal medium                                                                  PG: PridhamGottlieb's inorganic medium, supplemented with 0.1% yeast          extract                                                                       Lm: Luedemann's organic medium                                                Incubation for 2 weeks at 37° C.                                  

It is to be understood that production of the BBM-928 complex of thepresent invention is not limited to the particular actinomycetes speciesstrain No. G455-101 described by the above growth and microscopiccharacteristics. These characteristics are given for illustrativepurposes only and the invention contemplates use of strains or mutantsproduced from the above-described organism by conventional means knownto the art such as exposure to X-ray radiation, ultraviolet radiation,nitrogen mustard, phage exposure, and the like, which are capable ofproducing the BBM-928 complex or individual components thereof.

PREPARATION OF ANTITUMOR ANTIBIOTIC BBM-928 COMPLEX

The process of the present invention for producing the antitumorantibiotic BBM-928 complex comprises cultivating by fermentationactinomycetes strain No. G455-101 in an aqueous solution containing anassimilable carbon source and an assimilable nitrogen source undersubmerged aerobic conditions until substantial antitumor antibioticactivity is imparted to said solution. Conventional fermentation methodsare employed in cultivating actinomycetes strain No. G455-101. Mediawhich are useful for the production of the antibiotic antitumor agentsof the instant invention include n assimilable source of carbon such asstarch, glucose, dextrin, maltose, lactose, sucrose, fructose, mannose,molasses, glycerol and the like. The nutrient medium should also containan assimilable nitrogen source such as protein, protein hydrolysate,polypeptides, amino acids, corn steep liquor, casein, urea and the likeas well as nutrient inorganic salts which provide inorganic anions andcations such as potassium, sodium, ammonium, calcium, sulfate,carbonate, phosphate, chloride, nitrate, and the like.

In producing the BBM-928 complex, any temperature conductive tosatisfactory growth of the organism may be employed. Temperaturesranging from about 20° to 45° C. are operable with a preferredtemperature for optimizing growth of the organism ranging from about 28°to 34° C. with a temperature range of 30° to 32° most preferred. Maximumproduction of the BBM-928 complex is generally obtained in about 4 to 6days. Conventional methods are employed in the fermentation period. Forexample, preparation of small amounts is conveniently carried out inshake flasks or by surface cultures. Preparation of large amounts ispreferably carried out under submerged aerobic culture conditions insterile tanks. With tank fermentation a vegetative inoculum is firstproduced in a nutrient broth by innoculating the broth culture with aspore from the organism to provide a young active seed culture which isthen aseptically transferred to the fermentation tank medium. Aerationin tanks and bottles may be provided by forcing sterile air through oronto the service of the fermenting medium with further agitation intanks provided by a mechanical impeller. Anti-foaming agents such assilicone oil, soybean oil and lard oil may be added as needed.

Antibiotic levels in the fermentation broth or the extracts of BBM-928complex can be determined by paper disc agar-diffusion assay usingSarina lutea as a test organism and employing nutrient agar as the assaymedium. The pH is adjusted 9.0 for optimal sensitivity of the assaysystem which is used to determine optimum broth potency.

The BBM-928 complex is isolated from the fermentation broth byconventional means such as solvent extraction procedures. Purificationis conveniently carried out by preparative countercurrent distributionand chromatographic procedures as more fully described in Examples 2 and3 below to provide BBM-928 components A, B, C, D, E, and F.

PHYSICO-CHEMICAL PROPERTIES OF BBM-928 COMPONENTS A, B, C, AND D OFEXAMPLE 3

Individual components of BBM-928 show solubility and color reactionssimilar to each other. For example, they are readily soluble inchloroform and methylene chloride, slightly soluble in benzene, ethanol,methanol and n-butanol and insoluble in water and n-hexane. Positivereactions with ferric chloride and Ehrlich reagents are obtained withnegative reactions to Tollens, Sakaguchi and ninhydrin.

Characteristic physico-chemical properties of BBM-928 components ofExample 3 are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Physico-chemical Properties of                                                BBM-928 Components A, B, C and D                                                         BBM-928                                                                       A      B        C        D                                         ______________________________________                                        melting point                                                                              246-     214-     244-   224-                                                 248° C.                                                                         217° C.                                                                         248° C.                                                                       227° C.                          [α].sub.D.sup.25 (c 1, CHCl.sub.3)                                                   -27°                                                                            -74°                                                                            -91°                                                                          -13°                             Anal. found                                                                             C:     53.19    50.14  51.77  50.75                                           H:      5.40     5.29   5.29   5.25                                           N:     12.92    12.34  13.55  12.58                                 (by difference) O:                                                                      28.49  32.23    29.39  31.42                                        λ.sub.max in nm (E.sup.1% .sub.1 cm)                                   in EtOH      235(586) 235(570) 235(638)                                                                             235(550)                                             264(415) 264(400) 264(442)                                                                             264(380)                                             345(165) 345(163) 345(173)                                                                             345(155)                                in EtOH--HCl 234(610) 234(556) 234(650)                                                                             234(565)                                             264(410) 264(446) 264(442)                                                                             264(405)                                             345(165) 349(188) 345(173)                                                                             345(165)                                in EtOH--NaOH                                                                              230(564) 230(530) 230(580)                                                                             230(650)                                             256(763) 256(775) 256(704)                                                                             256(930)                                             330(180) 330(116) 330(117)                                                                             330(140)                                             383(170) 383(122) 383(122)                                                                             383(145)                                Mol. wt. (Osmometer,                                                                       1,450    --       1,470  --                                      in CHCl.sub.3)                                                                ______________________________________                                    

Infrared (IR) and nuclear magnetic resonance (NMR) spectra of componentsA, B, C and D are shown in FIG. 1-4 and FIG. 5-8, respectively, of theaccompanying drawings. The NMR spectra of BBM-928A (FIG. 5), BBM-928B(FIG. 6), and BBM-928C (FIG. 7) are very similar to each other with theonly difference being the presence of acetyl groups in components A (δ:2.03 ppm, 2 mol equivalent) and B (δ: 2.05 ppm, 1 mol equivalent) butnot in C. Upon acetylation with acetic anhydride in pyridine, 2, 3 and 4molar equivalents of acetyl group were introduced to BBM-928 componentsA, B and C, respectively. The three acetylation products thus obtainedshowed identical properties in TLC, UV, IR and NMR spectra, indicatingthat BBM-928A is a monoacetyl derivative of BBM-928B and a diacetylderivative of BBM-928C.

Acid hydrolysis of BBM-928A afforded five n-butanol-soluble,UV-absorbing fragments (I, II, III, IV and V) and five water-soluble,ninhydrin-positive substances (NPS-1, 2, 3, 4 and 5). The lattersubstances were separated by Dowex 50×4 chromatography and identified asthe following amino acids:

    ______________________________________                                        Compounds                                                                             Rf (S-123)*                                                                             Identification                                              ______________________________________                                        NPS-1   0.72      β-hydroxy-N--methylvaline (HM-Val)                     NPS-2   0.49      glycine (Gly)                                               NPS-3   0.46      serine (Ser)                                                NPS-4   0.45      sarcosine (sar)                                             NPS-5   0.23      (Not assigned)                                              ______________________________________                                         *TLC, silica gel plate                                                        S123: 10% ammonium acetatemethanol-10% ammonia solution (9:10:1)         

The five UV-absorbing fragments referred to above are shown to have thefollowing structures: ##STR1##

Upon acid hydrolysis (6N HCl), Fragments I, II, III and V gave thefollowing degradation products:

    ______________________________________                                                  Hydrolysis condition                                                            sealed tube    refluxed                                           Fragment    110°, 20 hrs                                                                          110° C., 3 hrs                              ______________________________________                                         I          IV, Ser        --                                                 II          IV, Ser, HM-Val                                                                              I, HM-Val                                          III         IV             --                                                 V           --             I, HM-Val, Sar                                     ______________________________________                                         Ser: serine                                                                   HMVal: hydroxy-N--methylvaline                                                Sar: sarcosine                                                           

Base hydrolysis of BBm-928A or BBM-928C with 0.1N NaOH at 25° C. for 3hr. provides Fragment VI (abbreviations Ser, HM-Val, and Sar are asstated above with Gly representing glycine and the symbol "X"representing an unassigned moiety) ##STR2##

Treating Fragment VI with 0.1N HCl at 110° c. for 1 hr. providesFragment VII plus HM-Val. ##STR3##

Treating Fragment VI with 0.1N NaOH at 37° C. for 40 Hr. providesFragment VIII plus Fragment I.

    X→Gly→Sar→HM--Val                     Fragment VIII

Treating Fragment VII with 0.1N NaOH at 37° C. for 40 hr. providesFragment IX plus Fragment I.

    X→Gly→Sar                                    Fragment IX

The unassigned moiety (X) has a molecular formula of C₅ H₆ N₂ O₂ (inpeptide form) based on microanalysis of Fragment IX and other peptidefragments containing (X) and spectral analysis summarized below.

    ______________________________________                                        .sup.13 CNMR                                                                             Proton-NMR    Assignment                                           ______________________________________                                         30.14 (t) 2.36 ppm (2H, m)                                                                            CH.sub.2                                              ##STR4##   4.2-4.5 (2H, m)                                                                             ##STR5##                                            140.7 (d)  6.76 (1H, t)  CHN                                                  171 (s)    --            CO (amide)                                                                    OH                                                                            NH                                                   ______________________________________                                    

According to spectral data for Fragments VIII and IX and a 360 MHzproton NMR to BBM-928A_(p) of Example 4, the unassigned amino acidmoiety (X) appears to be best respresented by the followingtetrahydropyridazine structure: ##STR6##

Based on the results of the above-described degradation experiments,spectral data, microanalysis and molecular weight determinations, thefollowing structures are believed to best represent BBM-928A, B and C:

    __________________________________________________________________________     ##STR7##                                                                                                      R.sub.1        R.sub.2                       __________________________________________________________________________                 BBM-928 A           Ac             Ac                                         BBM-928 B           Ac             H                                          BBM-928 C           H              H                             __________________________________________________________________________     Ser: serine                                                                   Gly: glycine                                                                  Sar: sarcosine                                                                HMVal: β-hydroxyNmethylvaline                                       

ANTIMICROBIAL ACTIVITY

Antimicrobial activity of BBM-928 components was determined against avariety of bacteria and fungi by serial agar dilution method in nutrientagar at pH 7 using Steer's multi-inoculating apparatus. The inoculumsize was standardized to apply a 0.0025-ml aliquot of test organismscontaining approximately 10⁴ cell/ml for all bacteria and fungi exceptacid-fast bacteria for which a 10⁶ cell/ml suspension was used. Minimuminhibitory concentrations (MIC), determined after overnight incubationat 37° C., are shown in Table 5. As seen therein, BBM-928 components aremoderately to weakly active against gram-positive and acid fast bacteriabut practically inactive against gram-negative bacteria and fungi.

The activity of prophage induction in lysogenic bacterium (ILB) wasdetermined for BBM-928 components. No significant ILB activity wasdemonstrated with BBM-928 components A, B and C up to a concentration of100 mcg./ml.

                                      TABLE 5                                     __________________________________________________________________________    In Vitro antimicrobial Activity of BBM-928 Components Against Aerobic         Bacteria                                                                      BBRI           BBM-928 Components (MIC in mcg/ml)                             Code                                                                              Test Organism                                                                            A     B     C     D     E     F                                __________________________________________________________________________    Sa-1                                                                              S. aureus 209P                                                                           12.5  25    50    100   100   25                               Sp-1                                                                              S. pyogenes S-23                                                                         6.3   12.5  25    50    50    12.5                             Sl-1                                                                              S. lutes PCI 1001                                                                        6.3   12.5  50    50    50    25                               Mf-1                                                                              M. flavus D12                                                                            12.5  25    50    100   100   25                               Cr-1                                                                              C. xerosis 53K-1                                                                         25    50    >100  >100  >100  50                               Bs-1                                                                              B. subtilis PCI 219                                                                      25    25    100   100   100   25                               Bg-1                                                                              B. megaterium D2                                                                         25    25    50    100   100   25                               Ba-3                                                                              B. anthracis A9504                                                                       6.3   12.5  25    50    50    12.5                             M6-1                                                                              M. smegmatis 607 D87                                                                     25    25    25    100   100   25                               Mp-1                                                                              M. phlei D88                                                                             12.5  12.5  12.5  50    50    12.5                             Ec-1                                                                              E. coli NIHJ                                                                             >100  >100  >100  >100  >100  >100                             Kp-1                                                                              K. pneumoniae D-11                                                                       >100  >100  >100  >100  >100  >100                             Pa-3                                                                              P. aeruginosa A9930                                                                      >100  >100  >100  >100  >100  >100                             Pv-1                                                                              P. vulgaris A9436                                                                        >100  >100  >100  >100  >100  >100                             Pm-1                                                                              P. mirabilis A9554                                                                       >100  >100  >100  >100  >100  >100                             Pg-1                                                                              P. morganii A9553                                                                        >100  >100  >100  >100  >100  >100                             Sm-1                                                                              S. marcescens A20019                                                                     >100  >100  >100  >100  >100  >100                             Al-1                                                                              A. faecalis ATCC 8750                                                                    >100  >100  >100  >100  >100  >100                             Ca-1                                                                              C. arbicans IAM 4888                                                                     >100  >100  >100  >100  >100  >100                             Cn-3                                                                              C. neoformans                                                                            100   >100  100   >100  >100  >100                             __________________________________________________________________________

ANTITUMOR ACTIVITY

Comparative testing of BBM-928 components A, B, C and D with mitomycin Cfor antitumor activity was carried out with the intraperitoneallyimplanted tumors: P388 leukemia, L1210 leukemia, B16 melanoma, LewisLung (LL) carcinoma, and sarcoma 180 ascites (S180). Test solutions ofthe BBM-928 components in 0.9% saline containing 10% dimethylsulfoxideand mitomycin C in 0.9% saline were administered once a day according todosing schedules ranging from a single one-day treatment to multipledaily treatments. By varying dosage, the minimum effective dose (MED)was determined which provided a median survival time value for thetreated animals at least 1.25 times greater than a control group. Thislevel of activity is considered to be a measure of significant antitumoractivity. Results are shown in Table 6 along with calculated activityratios illustrating that BBM-928A is markedly more active than mitomycinC by a factor of 10 to 300 depending upon the tumor strain and dosingschedule. Intraperitoneal LD₅₀ values of BBM-928 components A, B, C andD and mitomycin C determined by the method of Van der Waerden, Arch.Expt. Path. Pharmak., 195, 389 (1940), are also shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Antitumor Activity and Toxicity of                                            BBM-928 A, B, C and D and Mitomycin C                                         Minimum Effective Dose      LD.sub.50, i.p.                                   (MED, mg/kg/day)            (mg/kg/                                           P388        L1210   B16     LL    S180  day)                                  ______________________________________                                        Treatment.sup.a                                                               BBM-928A                                                                              0.003   >0.1    0.003 0.03  0.003 0.13                                BBM-928B                                                                              0.1     --      --    --    --    0.18                                BBM-928C                                                                              --      --      --    --    --    0.81                                BBM-928D                                                                              0.003   --      --    --    --     0.083                              Mitomy- 0.1     3       1     0.3   0.3   9.3                                 cin C                                                                         Treatment.sup.b                                                               BBM-928A                                                                              0.001   0.003   0.003 0.003  0.0001                                                                              0.013                              Mitomy- 0.1     0.3     0.3   0.3   0.03  1.4                                 cin C                                                                         Activity Ratio (BBM-928A vs. Mitomycin C)                                     Treatment.sup.a                                                                       30      --      300   10    100                                       Treatment.sup.b                                                                       100     100     100   100   100                                       ______________________________________                                         .sup.a single treatment on day 1.                                             .sup.b daily treatments from day 1 to day 9.                             

EXAMPLE 1 Production of BBM-928 Complex

Agar Fermentation

A well-grown agar slant of a strain of Actinomycetes species G455-101 isused to inoculate vegetative medium containing 2% soluble starch, 1%glucose, 0.5% Pharmamedia, 0.5% yeast extract, 0.5% NZ-amine (Type A)and 0.1% CaCO₃ with the pH being adjusted to 7.2 before sterilization.The seed culture is incubated at 32° C. for 72 hours on a rotary shaker(250 rpm), and 5 ml. of the growth is transferred to a 500 ml.Erlenmeyer flask containing 100 ml. of fermentation medium composed of2% soluble starch, 1% Pharmamedia, 0.003% ZnSO₄.7H₂ O and 0.4% CaCO₃.The production of the BBM-928 complex generally reaches maximum afterabout 5 days shaking culture.

Tank Fermentation

A seed culture is shaken for 4 days in Erlenmeyer flasks and inoculatedto 100 liters of germination medium composed of 2.0% oat meal (QuakerProducts, Australia), 0.5% glucose, 0.2% dry yeast, 0.0008% MnCl₂.4H₂ O,0.0007% CuSO₄.7H₂ O, 0.0002% ZnSO₄.7H₂ O and 0.0001% FeSO₄.7H₂ O in a200 liter seed tank fermentor which is stirred at 200 rpm. at 30° C. for54 hours. A 15-liter portion of the seed culture is then inoculated to170 liters of fermentation medium containing 2.0% soluble starch, 1.0%Pharmamedia, 0.003% ZnSO₄.7H₂ O and 0.4% CaCO₃ in a 400-liter tankfermentor which is operated at 30° C. at 200 rpm with an aeration rateof 150 liters/min. The broth pH gradually increases with the progress offermentation and reaches 8.4-8.5 after 100-120 hours at which time apeak antibiotic potency of 30 mcg/ml is obtained.

EXAMPLE 2 Isolation of BBM-928 Complex by Solvent Extraction

Harvested broth (170 liters, pH 8.5) from Example 1 is filtered with aclarifying agent. Activity is found in both the mycerial cake andfiltrate. The mycerial cake is extracted twice with a solvent mixture ofacetone and methanol (1:1, 30 liter×2). Extracts are combined andevaporated under reduced pressure to give an aqueous concentrate whichis extracted with n-butanol. The broth filtrate is extracted twice withn-butanol (40 liter×2). Concentration of the combined n-butanol extractsunder reduced pressure and lyophylization of the residue provides acrude solid (21.4 g.). According to thin layer chromatography assay,this material is a complex consisting of three major components, A, Band C, and three minor components, D, E and F having Rf values as setforth in Table 7 below.

                  TABLE 7                                                         ______________________________________                                        Silica gel TLC of BBM-928 Components                                                    Rf Values*                                                                    System N-118**                                                                          System N-103***                                           ______________________________________                                        BBM-928A    0.71        0.48                                                  BBM-928B    0.53        0.26                                                  BBM-928C    0.27        0.07                                                  BBM-928D    0.73        0.53                                                  BBM-928E    0.56        0.34                                                  BBM-928F    0.39        0.17                                                  ______________________________________                                         *detection by UV scanner (Shimadzu CS910) at 345 nm                           **.sub.-n-butanol-methanol-water (63:27:10)                                   ***xylene-methylethylketone-methanol (5:5:1)                             

EXAMPLE 3 Purification of BBM-928 Complex

The crude complex of Example 2 is purified by a preparative countercurrent distribution apparatus (Mitamura, 100 ml/tube) using a solventsystem of carbon tetrachloride-chloroform-methanol-water (5:2:5:1).After 50 transfers, tube Nos. 5 through 20 are combined and concentratedto give pale yellow powder (4.4 g.) containing components A, B, C, D, Eand F. This mixture is dissolved in a small amount of chloroform andcharged on a column of silica gel C-200 (500 ml.) which is pretreatedwith ethyl acetate. The column is developed by ethyl acetate with anincreasing amount of methanol (2-5%, V/V) and fractions monitored byoptical density at 345 nm. Minor component D is eluted first with ethylacetate followed by component A. Components E, B and F are eluted nextin that order at 3% methanol concentration. Each fraction containing theappropriate component is evaporated under reduced pressure and theresidue crystallized from chloroform-methanol. Likewise, crudepreparation of component C is obtained from tube Nos. 21 through 35 ofthe above-described counter current distribution. Purification ofcomponent C is carried out by silica gel chromatography andcrystallization from chlorofrom-methanol. Yields for components A, B, C,D, E and F are, respectively, 998 mg., 420 mg., 848 mg., 130 mg., 119mg., and 114 mg.

EXAMPLE 4 Further Purification of Component BBM-928A of Example 3

Thin layer chromatography assay of the BBM-928A component of Example 3(employing a system consisting of 10% methanol in toluene) indicated thesame was not completely homogenous in that it contained additionalmaterial running just ahead of the BBM-928A component. The followingsteps were carried out to effect purification.

(1) The sample is chromatographed on silica gel using a linear gradientof chloroform to 6% methanol-chloroform. Fractions eluting between 2.4and 3.3% methanol-chloroform (containing the BBM-928A component plussome contaminant) are composited for the next step.

(2) A concave gradient is generated using three vessels containing 2%methanol toluene in the first two and 6% methanol-toluene in the third.The composite from Step 1 chromatographed (silica gel column) on thisgradient provides a minor component which elutes first closely followedby the purified BBM-928A component (referred to herein as BBM-928A_(p)).

Molecular weight of BBM-928A_(p), as determined by Field Desorption MassSpectrometry, is 1427 corresponding to an empirical formula of C₆₄ H₇₈N₁₄ O₂₄ (MW 1427.417).

    ______________________________________                                        Elemental Analysis (Samples dried at 100° C. for 18 hours).                           C     H       N       O.sup.a                                  ______________________________________                                        Calculated for C.sub.64 H.sub.78 N.sub.14 O.sub.24 :                                           53.85   5.51    13.74 26.90.sup.                             Found.sup.b :    52.47   5.48    13.81 28.24.sup.a                            ______________________________________                                         .sup.a By difference.                                                         .sup.b Average of three determinations.                                  

The physico-chemical properties of BBM-928D relative to those ofBBM-928A, B, and C are summarized in Tables 4 and 7. They are similarbut with differences in melting point, specific rotation, and Rf valuein the N-118 and N-103 solvent systems indentified in Table 7. Thegeneral spectral patterns of all four substances are very similarsignifying similar structures, but with certain notable differences inthe NMR spectra.

NMR spectrum of BBM-928D (FIG. 8) differs from that of BBM-928A (FIG. 5)only in the presence of one acetyl (δ: 2.06 ppm, s) and one propionyl(δ: 1.06, t and 2.33 ppm, q) group in BBM-928D and two acetyl signals(δ: 2.06 ppm, s) in BBM-928A.

Complete acid hydrolysis of BBM-928D (6N HCl, 100° C., 18 hrs.) in thefashion indicated above for BBM-928A afforded the same five hydrolysisproducts in equimolar ratio as were afforded by BBM-928A, viz.3-hydroxy-6-methoxy-quinaldic acid and the four amino acids glycine,sarcosine, D-serine, and L-β-hydroxy-N-methylvaline. The structuraldifference between BBM-928A and BBM-928D is therefore in amino acidmoiety X referred to above,(3S,4S)-4-hydroxy-2,3,4,5-tetrahydropyridazine-3-carboxylic acid, whichis present as the acetate ester in BBM-928A.

Upon mild alkaline hydrolysis (0.01N Na₂ CO₃, 25° C., 30 min.) BBM-928Dafforded BBM-928C, the non-acylated family member. Thus, BBM-928D is themonoacetyl-monopropionyl derivative of BBM-928C, that is the generalformula given above for BBM-928A, B, and C wherein for BBM-928D R₁ isacetyl and R₂ is propionyl; molecular formula C₆₅ H₈₀ N₁₄ O₂₄ differingfrom that of BBM-928A by one CH₂ -unit.

We claim:
 1. A biologically pure culture of the microorganismActinomadura sp. having each of the identifying characteristics of ATCCNo. 31491, and which is capable of producing the antitumor antibioticBBM 928 complex in recoverable quantity upon cultivation on an aqueousculture medium containing assimilable sources of carbon and nitrogenunder submerged aerobic conditions.